Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

Chris Szent-Gyorgyi, Robyn L. Stanfield, Susan Andreko, Alison Dempsey, Mushtaq Ahmed, Sarah Capek, Alan S. Waggoner, Ian A. Wilson, Marcel P. Bruchez

Research output: Contribution to journalArticlepeer-review

59 Scopus citations

Abstract

We report that a symmetric small-molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* fluorogen activating protein is a VL domain that binds malachite green (MG) dye to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two V L domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity-determining regions are spatially oriented to form novel V L/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high-affinity protein tags and capture reagents.

Original languageEnglish
Pages (from-to)4595-4613
Number of pages19
JournalJournal of Molecular Biology
Volume425
Issue number22
DOIs
StatePublished - Nov 15 2013

Keywords

  • cooperative binding
  • directed evolution
  • fluorogen activating protein
  • quaternary structure
  • yeast surface display

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